Phenotypic Switching in Mycoplasma gallisepticum Hemadsorption Is Governed by a High-Frequency, Reversible Point Mutation

Abstract

Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain R low , detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA ؊ variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA ؊ mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA ؉ clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.Mycoplasma gallisepticum is a round flask-shaped organism commonly inducing chronic respiratory disease in chickens (14,26,32) and infectious sinusitis in turkeys (7). Like a large number of other mycoplasmas, this avian pathogen colonizes its host via the mucosal surfaces of the respiratory tract and must adhere to the epithelial cells to withstand clearance by the host. This intimate contact is mediated by a bleb-like structure (27, 28), a unipolar terminal organelle that is similar to the tip structure of the two human pathogens, M. pneumoniae and M. genitalium also involved in adhesion to host cells. Both mycoplasma species were shown to enter epithelial cells (2, 13), and recent in vitro assays have revealed that M. gallisepticum is likewise capable of establishing intracellular residence in nonphagocytic eukaryotic cells (29). During infection of highly immunocompetent hosts, the ability to enter and survive within host cells may provide these mycoplasmas with a survival strategy that relies first on adhesion. Cytadhesins and related components have been extensively studied in M. pneumoniae (17), and the data emerging from similar studies in M. gallisepticum suggest the occurrence of a family of cytadhesin genes conserved among pathogenic mycoplasmas that colonize widely divergent hosts. The identification and the characterization of M. gallisepticum surface-exposed components with adhesive properties are therefore of major importance in understanding the factors involved in promoting successful infection.In recent years, a large collection of data has underlined the versatility of the mycoplasma surface a...

Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

“…In contrast to the stable GapA/CrmA-negative phenotype of R high , a reversible point mutation based on a base substitution at the beginning of the structural gapA gene was shown to govern a spontaneous, high-frequency phase variation in expression of gapA of RCL2 (15). This mutational event, which is distinct from that described above for R high , also has a polar effect on the transcription of crmA resulting in the concomitant phase variation of the CrmA product.…”

“…This mutational event, which is distinct from that described above for R high , also has a polar effect on the transcription of crmA resulting in the concomitant phase variation of the CrmA product. Oscillation in expression of the GapA and CrmA products is responsible for the variable binding of erythrocytes (hemadsorption [HA]) onto M. gallisepticum colonies (15). This was demonstrated using a lineage derived from RCL2 and composed of clonal variants that alternatively displayed the HApositive (HA ϩ ) and HA Ϫ phenotypes and expressed or did not express the two cytadhesins, respectively.…”

“…Indeed, a high, avirulent passage of strain R (R high ) that lacks both GapA and CrmA has a diminished capacity to adhere to MRC5 human cells (13) and is deficient in binding erythrocytes on colonies (15) compared to a low, virulent passage of the same strain (R low ). In R high , the lack of GapA and CrmA is due to an additional adenine residue in the 5= region of the structural gapA gene that results in a premature stop codon and has a negative polar effect on the transcription of the crmA gene located downstream (13).…”

“…Among these are the products of the genes gapA (or mgc1) (11,12) and crmA (or mgc3) (13,14), which have been shown in independent in vitro studies to be the two major cytadhesins of M. gallisepticum strain R (13,15).…”

“…For this purpose, the colonizing ability and the virulence of a well-characterized HA Ϫ variant, namely, RCL2, that belongs to the clonal lineage described above, lacks the GapA and CrmA products, and displays a base substitution in the gapA gene (15) were addressed in this study. Since the GapA and CrmA cytadhesins are believed to be important in infection, we postulated that a back-switching from HA Ϫ to HA ϩ might confer a selective advantage in vivo.…”